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Application of Zmcps gene to preparation of male sterile line of corn

A male sterile line and gene technology, applied in the field of genetic engineering, can solve the problem of not finding the Zmcps male sterile line, etc., and achieve the effect of maintaining a homozygous recessive state, good benefits, and eliminating gene drift.

Active Publication Date: 2021-02-23
JILIN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Since the Zmcps gene was cloned, there has been no report on the use of transgenic technology to create Zmcps male sterile lines

Method used

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  • Application of Zmcps gene to preparation of male sterile line of corn
  • Application of Zmcps gene to preparation of male sterile line of corn
  • Application of Zmcps gene to preparation of male sterile line of corn

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Object Fragment Cloning

[0023] 1) Extraction of plant total RNA

[0024] (1) Wrap the mortar, tweezers, medicine spoon and triangular flask with tin foil, and then put them into a dry heat sterilization box for sterilization;

[0025] (2) 2 days after corn earing, take 50-100 mg of fresh corn tassels in a mortar, add an appropriate amount of liquid nitrogen to grind thoroughly, and then put the powder into a siliconized 1.5 mL centrifuge tube;

[0026] (3) Add 1mL Trizol, invert 5 times, and place at room temperature for 5 minutes;

[0027] (4) Add 200 μL of chloroform, shake vigorously for 15 seconds, place at room temperature for 5 minutes, and centrifuge at 12,000×g for 15 minutes at 4°C;

[0028] (5) Pipette the supernatant into a new siliconized 1.5mL centrifuge tube, add 500μL isopropanol, mix well, place at room temperature for 10min, and centrifuge at 12,000×g for 15min at 4°C;

[0029] (6) Remove the supernatant, add 1 mL of 75% ethanol, wash the...

Embodiment 2

[0079] Example 2 Construction of plant expression vector pCAMBIA3300-35s-Zmcps

[0080] 1) Extraction of pMD19-T-Zmcps plasmid

[0081] For small amount extraction of plasmids, refer to the OMEGA Plasmid Small Size Extraction Kit:

[0082] (1) Take an appropriate amount of overnight cultured target bacteria solution in a 1.5mL centrifuge tube, centrifuge at 12,000×g for 1min and discard the filtrate;

[0083] (2) Add 250 μL of SolutionI / RNase to resuspend the pellet;

[0084] (3) Add 250 μL of Solution II, gently and fully turn over several times to fully lyse the bacteria;

[0085] (4) Add 350 μL of SolutionIII, turn over gently and fully several times, and centrifuge at 12,000×g for 10 minutes;

[0086] (5) Aspirate the supernatant from step (4) and transfer it to a preparation tube, centrifuge at 12,000×g for 1 min, and remove the liquid;

[0087] (6) Add 500 μL of Buffer HB, centrifuge at 12,000×g for 1 min, and remove the liquid;

[0088] (7) Add 700 μL of Wash Buffe...

Embodiment 3

[0100] Embodiment 3 pCAMBIA3300-35s-Zmcps transforms Agrobacterium

[0101] 1) CaCl 2 Agrobacterium tumefaciens Competent Cells

[0102] (1) From the YEP tablet (Rif R ,Str R ) to inoculate a single colony of fresh EHA105 in YEP liquid medium containing 50mg / L Str and 25mg / L Rif, and cultivate overnight at 28°C and 220rpm for 24-36h;

[0103] (2) Take 2ml of overnight activated bacterial solution in the logarithmic growth phase, inoculate it in 50mL liquid YEP, and culture the bacterial solution OD at 20°C 600 to about 0.4~0.6;

[0104] (3) Transfer the bacterial solution to a 50 mL sterile centrifuge tube pre-cooled with ice, bathe in ice for 30 min, centrifuge at 4,000×g for 10 min at 4°C to enrich the bacterial cells;

[0105] (4) Pre-cool 0.05M CaCl with 10mL ice 2 Suspended cells, ice-bathed for 30 minutes, centrifuged at 4,000×g for 10 minutes at 4°C to enrich the cells;

[0106] (5) Pre-cool 0.05M CaCl with 1mL ice 2 Resuspend the bacteria, store the prepared co...

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Abstract

The invention discloses an application of a Zmcps gene to preparation of a male sterile line of corn, and belongs to the technical field of gene engineering. The invention provides the application ofthe Zmcps gene to preparation of the male sterile line of the corn. A protein Zmcps influencing male fertility can effectively realize complete abortion of corn pollen so as to be used for male sterile seed production of the corn, the corn seed production purity is improved, and the seed production cost is reduced.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, and more specifically relates to the application of the Zmcps gene in preparing maize male sterile lines. Background technique [0002] In actual food production, male sterility is common in higher crops such as corn, rice, and wheat, but most of the corn sterile lines are not complete, and a large number of inbred line seeds are mixed in the hybrids, which reduces the quality of the seeds. severely affected. The male sterile line is a good material to ensure the purity of hybrids and reduce the cost of seed production. [0003] The utilization of heterosis is the main way of high yield of maize. However, hybrids can only be exploited when effective and economical methods of pollination control exist in maize to ensure cross-pollination and prevent self-pollination. Mechanisms of pollination control include mechanical, chemical and genetic methods. [0004] Mechanical methods of h...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/84A01H5/00A01H6/46
CPCC07K14/415C12N15/8289
Inventor 宋广树高嵩吕庆雪孙蕾周德龙
Owner JILIN ACAD OF AGRI SCI
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